Gene Profiling Mechanisms of Cell Density-Dependent Growth in Myeloid and Lymphoid Leukemia Cell Lines.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4420-4420
Author(s):  
Ikuo Murohashi ◽  
Noriko Ihara
Keyword(s):  
Gene Expression ◽  
Growth Factors ◽  
Cell Density ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Lymphoid Leukemia ◽  
Cultured Cell ◽  
Cell Densities

Abstract Abstract 4420 Normal hematopoietic stem cells have been shown to be maintained through interaction with their environmental niches, such as osteoblastic and endothelial ones. The growth of leukemic cells has been shown to be stimulated by environmental niches (paracrine growth) or by cell-to-cell interaction or excreted factors of leukemic cells (autocrine growth). The growth of myeloid (MO7-E and HL-60) and lymphoid (Raji, U-266, Daudi and RPMI-1788) leukemia cell lines cultured at various cell densities in serum free medium (Sigma H 4281) with 1% BSA was evaluated. The cells cultured at higher cell densities (cultured cell densities ≥a 105/ml) showed logarithmic linear increases in cell number, whereas those at lower cell densities (cultured cell densities □… 104/ml) ceased increasing cell number. Supernatants of myeloid leukemia cells stimulated the growth of autologous clonogenic cells, but not those of lymphoid leukemia cells. Neutralizing antibodies (Abs) against various hematopoietic growth factors failed to inhibit cell growth except for anti-VEGF, which significantly decreased HL-60 leukemia cell growth. To clarify the nature of the cultured cell density on the growth of leukemia cells, leukemia cells were cultured at higher cell density (group H, cultured cell densities of 106/ml) or at lower cell density (group L, cultured cell densities 104/ml). After culture of 3-, 6-, 10-, and 24-hr, cells were serially harvested and total cellular RNA was extracted. Gene transcript levels were determined by using Real-Time PCR. Gene transcripts examined in the present study were as follows: polycomb (Bmi1), Hox (HOXA7, HOXA9, HOXB2, HOXB4, Meis 1), Caudal-related (CDX2, 4), Mef2c, c-Myb, Wnt (Wnt 3a, Wnt 5a, β-Catenin, β-Catenin, N-Cadherin), Notch (Notch-1, -2, -3 and Jagged-1, -2), CKI (p14, p15, p16, p18, p21, p27, p57), growth factor (VEGF, IGF-1, -2, Ang-1, -2, SDF-1), growth factor receptor (Flt-1, KDR, neurophilin-1, IGF-1R, Tie-1, -2, CXCR4), and growth related (c-Myc, CyclinD1, Foxo3a) genes. p18 and p21 gene expression was higher in group L compared with group H in two and all five groups, respectively. In contrast, p14 gene expression was higher in group H compared with group L. Any of the p15, p16, p27 and p57 genes was deleted. VEGF gene expression levels at 1-3- hr culture were higher in group H compared with group L. HOX, Meis 1 and Mef2c gene expression levels at 1- to 10- hr culture were higher in group H compared with group L. At 24-hr cultures, transcripts of myeloid and lymphoid cell lines for Bmi-1, Wnt-3a, and β-Catenin were higher, and those of lymphoid cell lines for Notch 1, 2, and 3 were higher in group H compared with group L. Taken together, our present results favor the conclusions that genes related to growth factors and transcription factors are sequentially and differentially expressed through cell-to-cell interaction and excreted autocrine growth factors of leukemia cells. Disclosures: No relevant conflicts of interest to declare.

Molecular Mechanisms of Cell Density-Dependent Growth in Myeloid and Lymphoid Leukemia Cell Lines.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4122-4122
Author(s):  
Ikuo Murohashi ◽  
Noriko Ihara
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Cell Number ◽  
Leukemia Cells ◽  
Lymphoid Leukemia ◽  
Hematopoietic Stem ◽  
Cultured Cell ◽  
Leukemia Cell Lines ◽  
Cell Densities

Abstract Normal hematopoietic stem cells have been shown to be maintained through interaction with their environmental niches, such as osteoblastic and endothelial ones. The growth of leukemia cells has been shown to be stimulated by environmental niches (paracrine growth) or by cell-to-cell interaction and/or excreted factors of leukemia cells (autocrine growth). The growth of myeloid (MO7-E and HL-60) and lymphoid (Raji, U-266, Daudi and RPMI-1788) leukemia cell lines cultured at various cell densities in serum free medium (Sigma H 4281) with 1% BSA was evaluated. The cells cultured at higher cell densities (cultured cell densities of more than 105/ml) showed logarithmic linear increases in cell number, whereas those at lower cell densities (cultured cell densities of less than 104/ml) ceased increasing cell number. Supernatants of myeloid leukemia cells stimulated the growth of autologous clonogenic cells, but not those of lymphoid leukemia cells. Neutralizing antibodies (Abs) against various hematopoietic growth factors failed to inhibit cell growth except for anti-VEGF Ab, which significantly decreased HL-60 leukemia cell growth. In contrast, anti-TNF-α Ab significantly stimulated the growth of the HL-60 cells. To clarify the nature of the cultured cell density on the growth of leukemia cells, leukemia cells were cultured at higher cell densities (group H, cultured cell densities of 106/ml) or at lower cell densities (group L, cultured cell densities of 104/ml). After culture of 3-, 6-, 10-, and 24-hr, cells were serially harvested and total cellular RNA was extracted. Gene transcript levels were determined by using Real-Time PCR. Gene transcripts examined in the present study were as follows: Jagged-1, -1, Notch-1, -2, -3, Ang-1, -2, Tie-1, -2, Wnd3a, Wnd5a, β-Catenin, γ-Catenin, N-Cadherin, Cyclin D1, p16, p21, HOXA6, HOXA7, HOXA10, HOXB4, and Mef2c. At 24-hr cultures, transcripts of myeloid leukemia cell lines for Bmi-1, Wnt-3a, β-Catenin and γ-Catenin were higher, and those of lymphoid leukemia cell lines for Notch 1, 2, and 3 were higher in group H compared with group L. Transcript levels for Wnt5a were higher at 10-hr culture (HL-60 and Raji), those for HOXA7 at 30–10-hr (MO-7E, U-266 and Raji), and those for Mef2c at 3-hr (MO-7E, U-266 and Raji) in group H compared with group L. Taken together, our present results favor the conclusions that genes related to transcription factors and growth factors are sequentially and differentially expressed through cell-to-cell interaction of leukemia cells. The nature of the leukemia cell-to-cell interacrtion, which is related to the growth advantages of leukemia stem cells over normal hematopoietic stem cells, remains to be further clarified.

RASSF2 Functions As a Tumor Suppressor in t(8;21) AML Via Promotion of Apoptosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3590-3590
Author(s):  
Samuel A Stoner ◽  
Russell Dekelver ◽  
Miao-Chia Lo ◽  
Dong-Er Zhang
Keyword(s):  
Gene Expression ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Hematopoietic Cells ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Mrna Levels ◽  
Human Cd34 ◽  
Leukemia Cell Lines ◽  
Cd34 Cells

Abstract The t(8;21) chromosomal translocation is one of the most common chromosomal translocations associated with acute myeloid leukemia (AML), found in approximately 12% of de novo AML cases. The majority of these cases are classified as FAB-subtype M2 AML. The t(8;21) results in the stable fusion of the AML1 (RUNX1) and ETO (RUNX1T1) genes. The AML1-ETO fusion protein is composed of the N-terminal portion of AML1, which includes the DNA-binding Runt-homology domain, and nearly the full-length ETO protein. The primary accepted mechanism by which AML1-ETO promotes leukemia development is through the aberrant recruitment of transcriptional repression/activation complexes to normal AML1 target genes. Therefore, the identification of individual genes or biological pathways that are specifically disrupted in the presence of AML1-ETO will provide further molecular insight into the pathogenesis of t(8;21) AML and lead to the possibility for improved treatment for these patients. We identified RASSF2 as a gene that is specifically downregulated in (2-4 fold) in total bone marrow of t(8;21) patients compared to non-t(8;21) FAB-subtype M2 AML patients by analyzing publicly available gene expression datasets. Similarly, using a mouse model of t(8;21) AML we found Rassf2 mRNA levels to be nearly 30-fold lower in t(8;21) leukemia cells compared to wild-type Lin-Sca-cKit+ (LK) myeloid progenitors. Gene expression analysis by RT-qPCR in leukemia cell lines confirmed that RASSF2 mRNA levels are significantly downregulated (8-10-fold) in both Kasumi-1 and SKNO-1 t(8;21) cell lines as compared to a similar non-t(8;21) HL-60 cell line and to primary human CD34+ control cells. In addition, expression of AML1-ETO in HL-60 or CD34+ cells results in a decrease in RASSF2 mRNA expression, which further suggests that RASSF2 is a target for regulation by AML1-ETO. Assessment of published ChIP-seq data shows that AML1-ETO binds the RASSF2 gene locus at two distinct regions in both primary t(8;21) AML patient samples and in the Kasumi-1 and SKNO-1 cell lines. These regions are similarly bound by several important hematopoietic transcription factors in primary human CD34+ cells, including AML1, ERG, FLI1, and TCF7L2, implicating these two regions as important for the regulation of RASSF2 expression during blood cell differentiation. Overexpression of RASSF2 in human leukemia cell lines using an MSCV-IRES-GFP (MIG) construct revealed that RASSF2 has a strong negative effect on leukemia cell proliferation and viability. The overall percentage of GFP-positive cells in MIG-RASSF2 transduced cells markedly decreased compared to MIG-control transduced cells over a period of 14 days. This effect was primarily due to significantly increased apoptosis in the RASSF2 expressing cell populations. Similarly, we found that expression of RASSF2 significantly inhibits the long-term self-renewal capability of hematopoietic cells transduced with AML1-ETO in a serial replating/colony formation assay. AML1-ETO transduced hematopoietic cells were normally capable of serial replating for more than 6 weeks. However, AML1-ETO transduced cells co-expressing RASSF2 consistently had reduced colony number and lost their ability to replate after 3-4 weeks. This was due to a dramatically increased rate of apoptosis in RASSF2 expressing cells. RASSF2 is reported to be a tumor suppressor that is frequently downregulated at the transcriptional level by hypermethylation in primary tumor samples, but not healthy controls. Here we have identified RASSF2 as a target for repression, and demonstrated its tumor suppressive function in t(8;21) leukemia cells. Further insights into the molecular mechanisms of RASSF2 function in AML will continue to be explored. Disclosures No relevant conflicts of interest to declare.

Anti-Apoptotic Proteins, HSP90 and Activated STAT3 Contribute to Busulfan Resistance of Myeloid Leukemia Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3472-3472
Author(s):  
Borje S. Andersson ◽  
Ben C. Valdez ◽  
David Murray ◽  
Latha Ramdas ◽  
Marcos de Lima ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Treatment Modalities ◽  
High Dose ◽  
Hematopoietic Stem ◽  
Conditioning Treatment ◽  
Apoptotic Genes

Abstract Busulfan(Bu)-based chemotherapy is a conditioning treatment prior to hematopoietic stem cell transplantation (HSCT) of patients with acute and chronic myelogenous leukemia (AML, CML). A major obstacle to successful HSCT is Bu resistance, which might be attributed to individual differences in drug pharmacokinetics and metabolism, or inherent resistance of cancer cells. We hypothesize that gene expression profiling of leukemia cells can be used to dissect the factors that contribute to their Bu resistance. Two Bu-resistant leukemia cell lines were established, characterized and analyzed by microarray and real-time RT-PCR techniques to identify differentially expressed genes. The CML B5/Bu2506 cells are 4.5-fold more resistant to Bu than their parental B5 cells. The AML KBM3/Bu2506 cells are 4.0-fold more Bu-resistant than KBM3 parental cells. Both resistant sublines evade Bu-mediated G2-arrest and apoptosis with constitutively up-regulated anti-apoptotic genes (BCL-XL, BCL2, BCL2L10, BAG3 and IAP2/BIRC3) and down-regulated pro-apoptotic genes (BIK, BNIP3, and LTBR).). Bu-induced apoptosis is partly mediated by activation of caspases; use of the inhibitor Z-VAD-FMK completely abrogated PARP1 cleavage and reduced apoptosis by ∼ 50%. Furthermore, Bu resistance in these cells may be attributed in part to up-regulation of HSP90 protein and activation of STAT3. Inhibition of HSP90 with geldanamycin attenuated phosphorylated STAT3 and made B5/Bu2506 and KBM3/Bu2506 more Bu-sensitive. Analysis of cells derived from patients classified as either clinically resistant or sensitive to high-dose Bu-based chemotherapy had alterations in gene expression that were analogous to those observed in the in-vitro model cell lines, confirming the potential clinical relevance of this model for Bu resistance. Our results suggest the important roles of apoptotic signaling mechanism, HSP90 and STAT3 and should be considered in the classification of AML patients who will likely benefit from busulfan-based pretransplant conditioning therapy and those who should be offered alternative treatment modalities.

Study on Leukemia Cell Differentiation and Drug Resistance By SATB1 Gene

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5172-5172
Author(s):  
Chunyan Wang ◽  
Huo Tan ◽  
Liu Lei ◽  
Lihua Xu
Keyword(s):  
Gene Expression ◽  
Drug Resistance ◽  
Cell Differentiation ◽  
Western Blot ◽  
Cell Lines ◽  
Jurkat Cell ◽  
Leukemia Cell ◽  
Jurkat Cells ◽  
Leukemia Cells ◽  
Chemotherapy Drugs

Abstract Background and objective: In this article, four types of lymphoma cell lines U937, Raji, Hut-102, Akata, and three types of leukemia cell lines K562, HL-60, Jurkat were used the expression of SATB1 in leukemia cells, Through cell differentiation induced by all-TRANS Retinoic acid and DMSO, finds the SATB1 gene expression is reduced, suggest that the SATB1 gene may be involved in cell differentiation. By gene silencing, reduce the SATB1 gene expression, sensitivity to chemotherapy drugs is increased, suggest that the SATB1 gene may be associated with drug resistance. Methods: 1. Western Blot Assay for detection of SATB1 gene expression in cell lines. 2. DMSO and ATRA to HL-60, Jurkat cells for different times, SATB1 gene expression in the cells are detected by Western Blot. 3. Building SATB1-shRNA, transfected Jurkat cells and to HL-60. 4. Testing inhibition rate of chemotherapy drugs on HL-60, HL-60-SATB1-ctr, HL-60-SATB1-sh, Jurkat, Jurkat-SATB1-ctr, Jurkat-SATB1-sh cell, suggesting its relationship with the drug resistance. Results: 1. SATB1 gene was expression in all the plant cells. 2. The expression of SATB1 in U937, Raji, Hut-102, Akata four types of lymphoma cell line is low. 3. It was highly expression in HL-60, Jurkat cells, but low in K562. 4. DMSO and ATRA treat HL-60, Jurkat cells for 48 and 96 hours, HL-60 cells become larger, rounded, Jurkat cells into smaller, is more obvious for 96-hour. 5. For HL-60, Jurkat cells, after DMSO and ATRA treated, the expression of SATB1 was decreased, more obvious for 96-hour. 6. HL-60-SATB1-sh1, Jurkat-SATB1-sh1 cell compared with the control group, the SATB1 protein content decreased significantly, successfully building SATB1-shRNA HL-60 and Jurkat cell line. 7. HL-60-SATB1-sh1, and Jurkat-SATB1-sh1 of daunorubicin (DNR) sensitivity has improved significantly. Conclusion: 1. The SATB1 gene expression in leukemia and lymphoma cells, and higher expression in leukemia cells. 2. After DMSO and ATRA treatment, the SATB1 expression significantly reduced, suggest that SATB1 may be involved in cell differentiation. 3. Successfully built SATB1-shRNA, and successfully transferred to HL-60 and Jurkat cell lines. 4. HL-60-SATB1-sh1, and Jurkat-SATB1-sh1 of daunorubicin (DNR) sensitivity increased significantly, prompting SATB1 may be related to drug-resistance. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Effect of Furanocoumarin Derivatives on Induction of Apoptosis and Multidrug Resistance in Human Leukemic Cells

Molecules ◽  
2019 ◽  
Vol 24 (9) ◽  
pp. 1824 ◽  
Author(s):  
Tomasz Kubrak ◽  
Marcin Czop ◽  
Przemysław Kołodziej ◽  
Marta Ziaja-Sołtys ◽  
Jacek Bogucki ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Annexin V ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Ic50 Values ◽  
Induction Of Apoptosis ◽  
Presence And Absence

Background: The insensitivity of cancer cells to therapeutic agents is considered to be the main cause of failure of therapy and mortality of patients with cancer. A particularly important problem in these patients is the phenomenon of multidrug resistance, consisting of abnormal, elevated expression of transport proteins (ABC family). The aim of this research included determination of IC50 values of selected furanocoumarins in the presence and absence of mitoxantrone in leukemia cells and analysis of changes in apoptosis using anexinV/IP and Casp3/IP after 24 h exposure of cell lines to selected coumarins in the presence and absence of mitoxantrone in IC50 concentrations. Methods: Research was conducted on 3 cell lines derived from the human hematopoietic system: HL-60, HL-60/MX1 and HL-60/MX2. After exposure to coumarin compounds, cells were subjected to cytometric analysis to determine the induction of apoptosis by two methods: the Annexin V test with propidium iodide and the PhiPhiLux-G1D2 reagent containing caspase 3 antibodies. Results: All of the furanocoumarin derivatives studied were found to induce apoptosis in leukemia cell lines. Conclusions: Our results clearly show that the furanocoumarin derivatives are therapeutic substances with antitumor activity inducing apoptosis in human leukemia cells with phenotypes of resistance.

scholarly journals Antimetabolic Cooperativity with l-Asparaginase and Tyrosine Kinase Inhibitor Quizartinib to Eradicate Persistant FLT3-ITD Leukemic Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 31-31
Author(s):  
Quentin Fovez ◽  
Raeeka Khamari ◽  
Anne Trinh ◽  
William Laine ◽  
Bruno Quesnel ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Glutamine Metabolism ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Metabolomic Analysis ◽  
Leukemia Cell Lines ◽  
Acute Myeloid

Introduction Acute myeloid leukemias are a group of malignant hemopathies characterized by a poor prognosis for survival. The discovery of oncogenic mutations in the FLT3 gene (eq FLT3-ITD) has led to the development of new tyrosine kinase inhibitors such as quizartinib. But complete remissions of patients remains difficult because these new TKIs are not able to completely eradicate all leukemia cells. Residual leukemia cells persist during treatment with quizartinib and lead to the rapid emergence of drug-resistant leukemia. Since mitochondrial oxidative metabolism supports the survival of leukemia cells after exposure to several anticancer drugs, we characterized the metabolism of leukemia cells that persisted within quizartinib treatment and developed metabolic strategies to eradicate them. Results First, we evaluated glycolysis activity in FLT3-ITD leukemia cell lines (MOLM13 / MOLM14 / MV4-11) under quizartinib treatment (5-10nM). Quizartinib reduced extracellular acidification rate ECAR, but this glycolytic activity is not fully inhibited (50% of untreated condition). These results obtained using the XFe24 Seahorse were in agreement with the metabolomic analysis carried out in a medium containing isotopic U-13C6 glucose. Next we evaluated mitochondrial oxidative phosphorylation in FLT3-ITD leukemia cell lines. After treatment with quizartinib, the basal and maximal oxygen consumption (OCR) of leukemia cells decreased. Metabolomic analysis using isotopic glucose U-13C6 or glutamine U-13C5 have shown that pyruvate derived from glucose was weakly oxidized in the mitochondria of untreated or quizartinib-treated cells. In contrast, a large amount of glutamine was oxidized by the tricarboxylic acid (TCA) cycle in untreated FLT3-ITD cells. Quizartinib reduced but did not abolish the complete oxidation of glutamine in leukemia cells. This result showed that even in the presence of quizartinib, FLT3-ITD cells maintained partially oxygen consumption trough glutamine oxidation. L-asparaginases (Kidrolase, Erwinase) are enzymes capable of hydrolyzing amino acids such as asparagine and glutamine. These clinical drugs have been approved for the treatment of chronic lymphocytic leukemia (CLL) and pediatric acute myeloid leukemia. We have shown that L-asparaginases weakly induced cell death in FLT3-ITD leukemia cells. Interestingly, our isobologram analysis showed that L-asparaginase acted synergistically with quizartinib to induce apoptosis. To determine whether glutamine metabolism also promoted the persistence of AML under treatment with quizartinib, we treated MOLM13 with quizartinib for several days. After long-term treatment, the percentage of surviving cells (annexin-V negative) was less than 5%. These persistent cells were characterized by an increased mitochondrial membrane potential (Δψm) and mitochondrial ROS. After treatment with the combination of L-asparaginase and quizartinib, the percentage of persistent cells decreased drastically. The combination of L-asparaginase and quizartinib was also more effective than quizartinib alone in reducing the size and number of colonies of MOLM13 in a model based on the formation of leukemia colonies growing in methylcellulose. Conclusion Persistent leukemia cells that survive after exposure to FLT3 inhibitor quizartinib can be targeted by the clinical drug L-asparaginases. This metabolic strategy could reduce the emergence of leukemic cells resistant to quizartinib. Disclosures Kluza: Daiichi-Sankyo: Research Funding.

Loss of p73 gene expression in lymphoid leukemia cell lines is associated with hypermethylation

2001 ◽  
Vol 25 (6) ◽  
pp. 441-447 ◽  
Author(s):  
Mingli Liu ◽  
Takeshi Taketani ◽  
Rongsheng Li ◽  
Junko Takita ◽  
Tomohiko Taki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Lymphoid Leukemia ◽  
Leukemia Cell Lines ◽  
P73 Gene

Bortezomib Interacts Synergistically with Belinostat to Induce Apoptosis In Human Acute Myeloid and Lymphoid Leukemia Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3266-3266
Author(s):  
Yun Dai ◽  
Shuang Chen ◽  
Li Wang ◽  
Xin-Yan Pei ◽  
Lora Kramer ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Dose Effect ◽  
Leukemia Cells ◽  
Parp Cleavage ◽  
Lymphoid Leukemia ◽  
Acute Leukemias ◽  
Leukemia Cell Lines ◽  
Acute Myeloid

Abstract Abstract 3266 Previous studies have demonstrated interactions between histone deacetylase (HDAC) and proteasome inhibitors (PIs) in multiple myeloma, NHL, and CLL. However, exploration of this strategy in acute leukemias has been more limited. In this context, we have previously demonstrated that HDACIs activate the cytoprotective NF-κB pathway in acute myeloid leukemia (AML) cells, and that interruption of this process dramatically increases lethality. Such findings raise the possibility that PIs, which block degradation of the NF-κB-inhibitory protein IκBα, may act via an analogous mechanism in acute leukemias. Consequently, interactions between the clinically relevant pan-HDAC inhibitor belinostat (PXD-101) and the FDA-approved proteasome inhibitor bortezomib were evaluated in both continuously cultured cell lines and primary AML and acute lymphoid leukemia (ALL) samples. First, whereas each agent individually displayed only modest toxicity, co-treatment for 24 hr or 48 hr with low concentrations of bortezomib (3 - 5 nM) and belinostat (50 - 300 nM) led to pronounced increases in apoptosis in diverse human acute leukemia cell lines (e.g., AML, U937, HL-60, MV-4-11/Flt3-ITD; T-cell ALL, Jurkat; B-cell ALL, SEM). Interactions between these agents were determined to be synergistic by Median Dose Effect analysis. Significantly, equivalent interactions were observed in multiple primary AML (n = 4) and ALL (n = 3) blast specimens, while largely sparing normal CD34+ hematopoietic cells isolated from umbilical cord blood (n = 4), as determined by annexin V/PI, DiOC6, and/or 7-AAD uptake by flow cytometry. Western blot analysis demonstrated that co-exposure of primary leukemia blasts to bortezomib and belinostat resulted in marked increase in PARP cleavage, compared with each agent administrated alone. In addition, cell morphology exhibited classical features of apoptosis in primary acute leukemia blasts, but not in normal CD34+ cells, following combination treatment. Second, in both cell lines and primary blasts, administration of bortezomib resulted in accumulation of the phosphorylated (S32/S36) form of IκBα, accompanied by diminished belinostat-mediated hyperacetylation (K310) of RelA/p65. Bortezomib also blocked processing of the precursor p100 into the active p52, an event enhanced by co-treatment with belinostat. These results indicate that a regimen combining bortezomib and belinostat interrupts both canonical and non-canonical NF-κB signaling pathways in acute leukemia cells. Moreover, co-exposure to these agents diminished expression of NF-κB-dependent pro-survival proteins including Bcl-xL, XIAP, and SOD2, but not NF-κB-independent anti-apoptotic proteins such as survivin. Third, because the BH3-only Bcl-2 family pro-apoptotic protein Bim plays an important role in the lethality of PIs or HDACIs as single agents, the expression and functional role of Bim in bortezomib/belinostat interactions was examined. Notably, whereas treatment with marginally toxic concentrations of either agent alone clearly increased Bim protein levels, co-exposure of either leukemia cell lines or primary blasts to bortezomib and belinostat led to sharply increased Bim expression (particularly the BimEL isoform). Importantly, shRNA knock-down of Bim substantially attenuated lethality mediated by co-treatment with bortezomib and belinostat in both AML (U937) and ALL (Jurkat) cells, supporting the notion that up-regulation of Bim plays a critical role in anti-leukemic activity of the combination regimen. Lastly, exposure of cultured leukemia cells and primary blasts to belinostat ± bortezomib induced hyperacetylation of a-tubulin, indicating inhibition of HDAC6, a microtubule-associated deacetylase that regulates aggresome formation and cell survival in response to misfolded protein-induced stress. Together, these findings indicate that the regimen combining belinostat and bortezomib is highly active against human AML and ALL cells, including primary leukemic blasts, in association with perturbation in the balance between pro-survival (NF-κB-dependent) and pro-death (e.g., Bim) signals. They also suggest that this strategy warrants further attention in acute leukemias. Accordingly, a Phase I trial of belinostat and bortezomib in patients with refractory acute leukemia or MDS has recently been initiated. Disclosures: Off Label Use: Investigational use of belinostat and bortezomib.

PD0332991, a CDK4/6 Inhibitor, Has An Anti-Leukemic Activity Against Lymphoid Crisis of CML and Ph+ALL Even with T315I Mutation in BCR-Abl; in Vitro Analysis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1511-1511
Author(s):  
Atsushi Nemoto ◽  
Takeshi Inukai ◽  
Koshi Akahane ◽  
Hiroko Honna- Oshiro ◽  
Kumiko Goi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Lymphoid Leukemia ◽  
Cycle Progression ◽  
Leukemia Cell Lines ◽  
T315i Mutation

Abstract Abstract 1511 Since BCR-ABL plays a central role in cell cycle progression of Philadelphia-chromosome positive (Ph+) leukemia cells and CDK4/6 critically involves in G1-progression of cell cycle, we analyzed sensitivity of Ph+ leukemia cell lines to compounds that act as specific CDK4/6 inhibitors. H3-thymidine uptake assay showed that both PD183812 and CBC219476 significantly inhibited cell growth of Ph+ lymphoid leukemia cell lines (n=9) in comparison with Ph+ myeloid leukemia cell lines (n=7) and Ph- ALL cell lines (n=26). Thus, we next tested the anti-leukemic activity of PD0332991, a potent CDK4/6 inhibitor that is under phase II clinical study for solid tumor patients, and found that 8 of 9 Ph+ lymphoid leukemia cell lines showed extremely higher sensitivity to PD0332991; median IC50 was <25 nM. IC50 of Ph+ lymphoid leukemia cell lines was significantly lower than that of Ph+ myeloid cell lines (200 nM, n=7) and Ph-ALL cell lines (100nM, n=25). PD0332991 effectively dephosphorylated Rb protein (pRb), and subsequently induced G1 arrest on all of Ph+ lymphoid leukemia cell lines. Moreover, PD0332991 gradually induced cell death in 4 Ph+ lymphoid leukemia cell lines. Since CDK4/6 inhibitor acts depending on intact pRb, we analyzed protein and gene expression status of Rb. Of note, all Ph+ lymphoid leukemia cell lines expressed intact pRb except for one cell line that showed relative resistance to PD0332991. In contrast, pRb was almost undetectable in Ph+ myeloid cell lines in spite of comparable level of Rb gene expression, which might be mechanism for resistance to PD0332991. However, most of Ph- ALL cell lines had intact pRb expression in spite of their relative resistance to PD0332991, indicating that Rb status alone did not explain higher PD0332991-sensitivity of Ph+ lymphoid leukemia cell lines. Thus, we assumed that Ph+ lymphoid leukemia cells showed higher PD0332991-sensitivity probably because BCR-ABL regulates CDK4/6 expression for cell cycle progression. To clarify this assumption, we treated Ph+ lymphoid leukemia cell lines with imatinib and performed immunoblot analysis of cell cycle machineries such as CDKs, cyclines, and CDK inhibitors. Of note, CDK4 expression level was frequently downregulated by imatinib in Ph+ lymphoid leukemia cell lines. Moreover, imatinib-induced downregulation of CDK4 in Ph+ lymphoid leukemia cell line was abrogated by the addition of IL-7 and FLT3 ligand, which stimulated cell cycle progression of imatinib-treated Ph+ ALL cell line. LY294002, a PI3K inhibitor, but not U0126, a MAPK inhibitor, and AG490, an inhibitor for JAK/STAT pathway, efficiently downregulated CDK4 expression in Ph+ lymphoid leukemia cell lines. Gene expression level of CDK4 in Ph+ lymphoid leukemia cell lines was downregulated by imatinib, and lactastatin, an inhibitor of protein degradation, partially inhibited imatinib-induced downregulation of CDK4 protein in Ph+ lymphoid leukemia cell lines, indicating that BCR-ABL regulates CDK4 expression both in gene expression level and in protein degradation level. These findings indicated that Ph+ lymphoid leukemia cell lines showed higher sensitivity to PD0332991 since BCR-ABL induces cell cycle progression of Ph+ lymphoid leukemia cells by regulating CDK4 as one of downstream pathways. Accordingly, we tested if PD0332991 shows anti-leukemic activity in Ph+ lymphoid leukemia cells that have a T315I mutation of BCR-ABL. SU/SR is an imatinib-resistant Ph+ ALL cell line with T315I mutation (IC50 for imatinib >10 mM), which was established from SU-Ph2, an imatinib-sensitive Ph+ ALL cell line (IC50 for imatinib <0.1 mM), after long-term culture in the presence of gradually increasing concentration of imatinib. Of note, PD0332991 effectively dephosphorylated pRb and inhibited cell growth of both SU/SR and SU-Ph2. Our findings provide a rationale for efficacy of PD0332991 in the context of anti-leukemic therapy for lymphoid crisis of CML and Ph+ ALL patients even with T315I mutation in BCR-ABL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The U2AF homology motif kinase 1 (UHMK1) is upregulated upon hematopoietic cell differentiation

2017 ◽  
Author(s):  
Isabella Barbutti ◽  
João Agostinho Machado-Neto ◽  
Vanessa Cristina Arfelli ◽  
Paula de Melo Campos ◽  
Fabiola Traina ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Hematopoietic Cell ◽  
Leukemic Cells ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Induced Differentiation ◽  
Aberrant Expression ◽  
Leukemia Cell Lines

AbstractUHMK1 (KIS) is a nuclear serine/threonine kinase that possesses a U2AF homology motif and phosphorylates and regulates the activity of the splicing factors SF1 and SF3b155. Mutations in these components of the spliceosome machinery have been recently implicated in leukemogenesis. The fact that UHMK1 regulates these factors suggests that UHMK1 might be involved in RNA processing and perhaps leukemogenesis. Here we analyzed UHMK1 expression in normal hematopoietic and leukemic cells as well as its function in leukemia cell line.In the normal hematopoietic compartment, markedly higher levels of transcripts were observed in differentiated lymphocytes (CD4+, CD8+ and CD19+) compared to the progenitor enriched subpopulation (CD34+) or leukemia cell lines. UHMK1 expression was upregulated in megakaryocytic-, monocytic-and granulocytic-induced differentiation of established leukemia cell lines and in erythrocytic-induced differentiation of CD34+ cells. No aberrant expression was observed in patient samples of myelodysplastic syndrome (MDS), acute myeloid (AML) or lymphoblastic (ALL) leukemia. Nonetheless, in MDS patients, increased levels of UHMK1 expression positively impacted event free and overall survival.Lentivirus mediated UHMK1 knockdown did not affect proliferation, cell cycle progression, apoptosis or migration of U937 leukemia cells, although UHMK1 silencing strikingly increased clonogenicity of these cells. Thus, our results suggest that UHMK1 plays a role in hematopoietic cell differentiation and suppression of autonomous clonal growth of leukemia cells.

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